Pancreatic cancer biomarkers based on extracellular vesicles

Abstract

The present disclosure relates to methods and kits for diagnosing pancreatic cancer and monitoring disease load in patients diagnosed with pancreatic cancer, the method comprises the quantitative determination of the concentration of extracellular vesicles in a sample of a bodily fluid of a patient that are positive against one, two or three markers selected from the group consisting of thrombin-sensitive protein-2 (THBS2), placental alkaline phosphatase-like protein 2 (ALPPL2) and macrophage migration inhibition factor (MIF).

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 405,863, filed on September 12, 2022, entitled “Extracellular Vesicle-Based Pancreatic Cancer Biomarkers,” the entire disclosure of which is incorporated herein by this reference. Technical Field

[0003] Embodiments of the present disclosure relate to extracellular vesicle (EV)-derived protein markers THBS2, ALPPL2, and MIF for monitoring disease burden in patients with pancreatic cancer with increased sensitivity in both CA19-9 positive and negative patients. Background Art

[0004] Pancreatic cancer is an aggressive malignancy that is often undiagnosed in its early stages. Therefore, non-invasive, early, and accurate diagnosis is undoubtedly the "holy grail" of pancreatic cancer research. In particular, pancreatic ductal adenocarcinoma (PDAC) is often diagnosed too late to be effectively treated. It is expected that by 2020, PDAC will become the second leading cause of cancer death in the United States. Most PDAC patients are diagnosed in the late stages of the disease, and their tumors cannot be surgically removed, resulting in a 5-year overall survival rate of approximately 12%. The lack of early diagnosis makes it challenging to develop treatments to slow or reverse PDAC.

[0005] The classical strategy for early detection of biomarker development includes an initial retrospective phase of discovery and validation of tissue samples collected from individuals diagnosed with the disease and compared with controls. Currently, blood sugar antigen 19-9 (CA19-9), a sialyl Lewis A sugar antigen, is often used as a marker for monitoring disease burden in pancreatic cancer patients. CA19-9 is a tetrasaccharide that is highly expressed in advanced adenocarcinomas such as colon cancer, gastric cancer, and pancreatic cancer. It is known that it plays an important role in the intercellular recognition process. It is also a tumor marker for detecting pancreatic cancer. It is known that PDAC patients have elevated CA19-9 levels (hereinafter referred to as CA19-9 secretors). However, about 15% of pancreatic cancer patients are Lewis antigen-negative, and they do not secrete or secrete less CA19-9. Therefore, there is still a need for a test to monitor disease burden in pancreatic cancer patients who do not secrete CA19-9 or do not have elevated CA19-9 levels (hereinafter referred to as CA19-9 non-secretors). Summary of the invention

[0006] A combination of novel biomarkers and biomarkers for diagnosing pancreatic cancer and monitoring disease load is disclosed. In some aspects, the present disclosure relates to a method for diagnosing pancreatic cancer in a patient. In other aspects, the present disclosure relates to a method for monitoring disease load in a patient diagnosed with pancreatic cancer, including but not limited to monitoring the appearance, disappearance, regression or progression of lesions in a patient diagnosed with pancreatic cancer or undergoing pancreatic cancer screening. In other aspects, the present disclosure relates to a method for treating pancreatic ductal adenocarcinoma (PDAC) in a patient, including determining the persistence of pancreatic cancer treatment or the dosage of a pancreatic cancer therapeutic agent. In certain embodiments, the patient does not secrete CA19-9. In other embodiments, the patient secretes CA19-9.

[0007] The method includes quantitatively determining the amount of one, two or three markers in a serum, plasma or pancreatic ductal fluid sample, which is selected from: thrombospondin-2 (THBS2) positive extracellular vesicles, placental alkaline phosphatase-like protein 2 (ALPPL2) positive extracellular vesicles and macrophage migration inhibitory factor (MIF) positive extracellular vesicles. In some embodiments, the marker comprises THBS2. In other embodiments, the marker comprises MIF. In other embodiments, the marker comprises a combination of ALPPL2 and another marker. In some embodiments, the marker comprises (a) THBS2 and ALPPL2, (b) MIF and ALPPL2, (c) MIF and THBS2 or (d) THBS2, ALPPL2 and MIF. For some embodiments, the method further comprises correlating the amount of the marker with the size of a tumor associated with pancreatic cancer.

[0008] For a method of diagnosing pancreatic cancer or monitoring disease burden in a patient diagnosed with pancreatic cancer, the method comprises obtaining a sample from the patient; quantitatively determining the concentration of one or more selected markers in the sample; comparing the quantitatively determined concentration of the one or more selected markers with a healthy control value of the selected marker or a patient's previous concentration; and diagnosing the presence of pancreatic cancer or monitoring disease burden in the patient based on the comparison, wherein a concentration higher than the healthy control value or the patient's previous concentration indicates pancreatic cancer or an increase in disease burden. The sample obtained is selected from the group consisting of plasma, serum, and pancreatic ductal fluid. In some embodiments, the one or more markers used for comparison are selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF)-positive extracellular vesicles, or (ii) placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF-positive extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles, and IF-positive extracellular vesicles. Certain methods of monitoring the appearance, disappearance, regression, or progression of a disease in a patient diagnosed with pancreatic cancer or undergoing pancreatic cancer screening include obtaining a sample from the patient; quantitatively determining the concentration of one or more selected markers in the sample; comparing the quantitatively determined concentration of the one or more selected markers to a healthy control value or a patient's previous concentration of the selected marker; and based on the comparison, monitoring the appearance, disappearance, regression, or progression of the disease in a patient diagnosed with pancreatic cancer or undergoing pancreatic cancer screening.

[0009] Concentrations higher than healthy control values ​​or higher than previous concentrations in patients indicate the presence and progression of pancreatic cancer. The samples obtained are selected from: plasma, serum, and pancreatic ductal fluid. In some embodiments, the markers are selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF)-positive extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF-positive extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles, and IF-positive extracellular vesicles.

[0010] For certain methods of treating pancreatic cancer (such as PDAC) in a patient, the method includes obtaining a first sample from the patient before neoadjuvant therapy; quantitatively determining the concentration of one or more selected markers in the first sample; administering neoadjuvant therapy to the patient; and then obtaining a second sample from the patient after administering neoadjuvant therapy. The method further includes quantitatively determining the concentration of one or more selected markers in the second sample; comparing the quantitatively determined concentrations of one or more selected markers between the first sample and the second sample; and when the concentration of one or more selected markers in the second sample is reduced compared to the first sample, the patient's pancreatic tumor is removed, thereby treating the patient's pancreatic ductal adenocarcinoma. In some embodiments of treating pancreatic cancer, the method further includes obtaining a third sample from the patient after resecting the patient's pancreatic tumor; quantitatively determining the concentration of one or more selected markers in the third sample; and administering adjuvant therapy to the subject when the concentration of one or more selected markers in the third sample is increased compared to the second sample. Increased concentrations of one or more selected markers indicate an increased disease burden. The sample obtained is selected from: plasma, serum, and pancreatic ductal fluid. The marker is selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF)-positive extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF-positive extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles and IF-positive extracellular vesicles.

[0011] In some embodiments, the concentration of marker-positive extracellular vesicles (EVs) in a sample is quantitatively determined by using antibodies to selected markers, fluorescently labeling the markers to positive EVs, and determining the amount of EVs that are positive for one or more selected markers. In some aspects, the antibodies used are fluorescently labeled antibodies to selected markers. In specific embodiments, the concentration of marker-positive vesicles in a sample is quantitatively determined using the Nanoview EV assay. In some embodiments, it is determined by ELISA whether a patient secretes CA19-9. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The embodiments will be described below with reference to the drawings and / or included drawings, wherein like reference numerals refer to like elements.

[0013] Figure 1AShown are the concentrations of biomarker-positive EVs for placental alkaline phosphatase-like protein 2 (ALPPL2) and thrombospondin-2 (THBS2) in healthy controls and patients (CA19-9 non-secretors or secretors).

[0014] Figure 1B Shown are the concentrations of MIF-positive EVs in healthy controls and patients (CA19-9 non-secreters or secretors) and the concentrations of serum CA19-9 (determined by ELISA) in patients (CA19-9 non-secreters or secretors).

[0015] Figure 2A The concentrations of biomarker-positive EVs for ALPPL2 and THBS2 before and after treatment in CA19-9 secretors are shown.

[0016] Figure 2B Shown are the concentrations of MIF-positive EVs and serum CA19-9 before and after treatment in CA19-9 secretors.

[0017] Figure 3A Shown are the concentrations of biomarker-positive EVs for ALPPL2 and THBS2 in patients with normalization of CA19-9 after treatment.

[0018] Figure 3B Shown are the concentrations of MIF-positive EVs and serum CA19-9 in patients in whom CA19-9 normalized after treatment.

[0019] Figure 4A The concentrations of biomarker-positive EVs for ALPPL2 and THBS2 before and after treatment in CA19-9 non-secretors are shown.

[0020] Figure 4B Shown are the concentrations of biomarker-positive EVs before and after treatment against MIF and CA19-9 in CA19-9 non-secretors.

[0021] Figure 5A Baseline levels of concentrations of double-positive EVs in healthy controls and patients (CA19-9 non-secretors or secretors) are shown.

[0022] Figure 5B Shown are the concentrations of double-positive EVs for MIF and THBS2 before and after treatment in CA19-9 non-secretors or secretors.

[0023] Figure 5C Shown are the concentrations of MIF and THBS2 double-positive EVs before and after treatment in CA19-9 non-secretor patients.

[0024] Fig. 6A It was shown that dual combinations of MIF / APPL2 double-positive EVs or ALLP2 / THBS2 double-positive EVs were used to effectively monitor disease burden in PDAC patients (CA19-9 secretors).

[0025] Figure 6B The triple combination of MIF, ALPPL2 and THBS2 was shown to be effective for monitoring disease burden in PDAC patients (CA19-9 secretors).

[0026] Figure 6C The combination of AlPPL2 and THBS2 was shown to be effective in monitoring disease burden in PDAC patients (CA19-9 secretors).

[0027] Fig. 7A Shown is a correlation plot between the ALPP2 marker and tumor size (RECIST) in a single patient (CA19-9 secretor).

[0028] Figure 7B Shown is a correlation plot between THBS2 marker and tumor size (RECIST) in a single patient (CA19-9 secretor).

[0029] Figure 7C Shown is a correlation plot between MIF marker and tumor size (RECIST) in a single patient (CA19-9 secretor).

[0030] Fig.7D Shown is a correlation plot between CA19-9 marker and tumor size (RECIST) in a single patient (CA19-9 secretor).

[0031] Fig. 8A Shown is a correlation plot between the ALPP2 marker and tumor size (RECIST) in a single patient (CA19-9 non-secretor).

[0032] Figure 8B Shown is a correlation plot between THBS2 marker and tumor size (RECIST) in a single patient (CA19-9 non-secretor).

[0033] Figure 8C Shown is a correlation plot between MIF marker and tumor size (RECIST) in a single patient (CA19-9 non-secretor). DETAILED DESCRIPTION

[0034] Specific aspects and applications of the present disclosure are described in the following figures and detailed embodiments of the technology. Unless otherwise noted, words and phrases in the specification and claims should be given the clear, ordinary and customary meanings to those of ordinary skill in the applicable art.

[0035] In the following description, and for the purpose of explanation, many specific details are set forth in order to provide a thorough understanding of various aspects of the present disclosure. However, it will be appreciated by those skilled in the relevant art that the embodiments of the technology disclosed herein can be practiced without these specific details. For example, in order not to make the present disclosure unnecessarily obscure, known processes and production techniques are not particularly described in detail. It should be noted that the disclosed technology can be applied to many different and alternative configurations, devices and techniques. The full scope of the technology disclosed herein is not limited to the examples described below.

[0036] The present disclosure, its aspects and embodiments are not limited to the specific packaging types, material types or other system component examples or methods disclosed herein. Many additional components, production and assembly procedures consistent with semiconductor wafer manufacturing, production and packaging known in the art can be used for specific embodiments of the present disclosure. Thus, for example, although specific embodiments are disclosed, such embodiments and components of the embodiments may include any components, models, types, materials, versions, quantities, etc. for these systems and implementation components that are consistent with the intended operation known in the art.

[0037] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a step" includes reference to one or more of such steps.

[0038] The words "exemplary," "example," or various forms thereof, are used herein to indicate use as an example, instance, or illustration. Any aspect or design described herein as "exemplary" or "example" is not necessarily to be construed as preferred or superior to other aspects or designs. In addition, the examples provided are for purposes of clarity and understanding only and are not meant to limit or restrict the disclosed subject matter or relevant portions of the present disclosure in any way. It should be appreciated that additional or alternative examples of numerous different scopes could be presented, but these examples have been omitted for the sake of brevity.

[0039] When a range of values ​​is expressed, another embodiment includes from one specific value and / or to another specific value. Similarly, when values ​​are expressed as approximations, by using the antecedent "about", it is understood that the specific value forms another embodiment. All ranges are inclusive and can be combined.

[0040] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of these words, such as “comprising” and “comprises”, mean “including but not limited to”, and are not intended to (and do not) exclude other components.

[0041] As used herein, the term "neoadjuvant therapy" refers to chemotherapy administered to a patient before surgery and / or radiotherapy is used to treat a primary tumor. The term is a synonym for preoperative chemotherapy. In the context of pancreatic cancer, the primary therapy is usually tumor resection and the corresponding adjuvant therapy is chemotherapy. In the context of pancreatic cancer, the best neoadjuvant chemotherapy currently known uses FOLFIRINOX (a chemotherapy combination containing the drugs leucovorin (folinic acid), fluorouracil, irinotecan hydrochloride, and oxaliplatin), gemcitabine, alone or in combination with chemotherapeutic agents.

[0042] As used in this article, the term "adjuvant therapy" refers to the treatment given to the patient after the primary treatment, reducing the chance of cancer recurrence by destroying any remaining cancer cells. For patients with unresectable tumors or higher surgical risks, the primary therapy can be chemotherapy or radiotherapy. In the context of pancreatic cancer, primary therapy is usually tumor resection and corresponding adjuvant therapy is chemotherapy. For pancreatic cancer, the selection of current adjuvant chemotherapy includes but is not limited to FOLFIRINOX (chemotherapeutic combination containing medicine leucovorin calcium (folinic acid), fluorouracil, irinotecan hydrochloride and oxaliplatin), the combination of gemcitabine and albumin-bound paclitaxel (Nab-Paclitaxel), the combination of gemcitabine and capecitabine, the combination of gemcitabine and erlotinib or single gemcitabine.

[0043] As required, the specific embodiments of the present disclosure are included herein. It should be understood that the disclosed embodiments are merely examples of the present invention that can be embodied in various forms. Therefore, the specific structural and functional details disclosed herein should not be interpreted as limitations, but are merely the basis for teaching those skilled in the art to use the present invention. The following specific examples will enable the present disclosure to be better understood. However, they are provided only as guidance and do not imply any limitation.

[0044] By referring to the following detailed description in conjunction with the drawings and examples, the present disclosure can be more easily understood, and the drawings and examples form a part of the present disclosure. It should be understood that the present disclosure is not limited to the specific materials, devices, methods, applications, conditions or parameters described and / or shown herein, and the terms used herein are only used to describe specific embodiments by example, and are not intended to limit the claimed invention. As used in this article, the term "multiple" refers to more than one. When representing the range of values, another embodiment includes from a specific value and / or to another specific value. Similarly, when a value is expressed as an approximation, by using the antecedent "about", it is understood that a specific value forms another embodiment. All ranges are inclusive and can be combined.

[0045] The present disclosure relates to novel biomarkers and combinations of biomarkers for diagnosing pancreatic cancer and monitoring disease burden. The biomarkers described herein are thrombospondin-2 (THBS2) positive extracellular vesicles, placental alkaline phosphatase-like protein 2 (ALPPL2) positive extracellular vesicles, and macrophage migration inhibitory factor (MIF) double positive EV extracellular vesicles. ALPPL2 is a protein that is a diagnostic biomarker for pancreatic ductal adenocarcinoma and is not substantially expressed in normal tissue. THBS2 is a protein belonging to the family of matrix cell calcium-binding glycoproteins that has been found to be a biomarker for pancreatic cancer. MIF, also known as glycosylation inhibitory factor (GIF), L-dopamine isomerase, or phenylpyruvate tautomerase, is a protein encoded by the MIF gene, which is a regulator of innate immunity. In particular, the methods herein relate to the use of (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF) double-positive EV extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF double-positive EV extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles, and IF double-positive EV extracellular vesicles; to diagnose pancreatic cancer in patients, monitor disease burden (e.g., monitor the appearance, disappearance, regression, or progression of the disease) in patients diagnosed with pancreatic cancer, treat pancreatic cancer in patients, and / or monitor treatment effectiveness in patients diagnosed with pancreatic cancer. Uses include the production of diagnostic kits and drugs for pancreatic cancer. The disclosed biomarkers are useful for diagnosis, disease monitoring, and treatment monitoring of pancreatic tumors that secrete CA19-9 as well as pancreatic tumors that do not secrete CA19-9. Therefore, the disclosed biomarkers are useful even for suspected or confirmed pancreatic cancer cases where the CA19-9 secretion status is unknown.

[0046] Methods for diagnosing pancreatic cancer in a patient, monitoring disease burden in a patient diagnosed with pancreatic cancer, treating pancreatic cancer in a patient diagnosed with pancreatic cancer, and / or monitoring treatment effectiveness include quantitatively determining the amount of the aforementioned biomarkers or combinations of biomarkers in a serum, plasma, or pancreatic ductal fluid sample. In some embodiments, the concentration of marker-positive vesicles in the sample is quantitatively determined by separating one or more selected markers using antibodies to the selected markers and determining the amount of the separated one or more selected markers. In some aspects, the antibody used is a fluorescently labeled antibody to the selected marker. In certain embodiments, the quantitative determination is performed by the NanoView EV assay. For some embodiments, the method further includes correlating the amount of the marker with the size of the tumor associated with pancreatic cancer.

[0047] For a method of diagnosing pancreatic cancer or monitoring disease burden in a patient diagnosed with pancreatic cancer, the method comprises obtaining a sample from the patient; quantitatively determining the concentration of one or more selected markers in the sample; comparing the quantitatively determined concentration of EVs positive for one or more selected markers with a healthy control value or a patient's previous concentration of EVs positive for the selected markers; and diagnosing the presence or absence of pancreatic cancer or monitoring disease burden in the patient based on the comparison, wherein a concentration higher than a healthy control value or higher than a patient's previous concentration indicates pancreatic cancer or increased disease burden. The sample obtained is selected from the group consisting of: plasma, serum, and pancreatic ductal fluid. The one or more markers used for comparison are selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF) double-positive EV extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF double-positive EV extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles and IF double-positive EV extracellular vesicles.

[0048] Certain methods of monitoring the appearance, disappearance, regression, or progression of disease in a patient diagnosed with or being screened for pancreatic cancer include obtaining a sample from the patient; quantitatively determining the concentration of EVs that are positive for one or more selected markers in the sample; comparing the quantitatively determined concentration of EVs that are positive for one or more selected markers to a healthy control value or the patient's previous concentration of EVs positive for the selected markers; and monitoring the appearance, disappearance, regression, or progression of disease in the patient diagnosed with or being screened for pancreatic cancer based on the comparison.

[0049] Concentrations higher than healthy control values ​​or previous concentrations of the patient indicate the presence and progression of pancreatic cancer. The samples obtained are selected from: plasma, serum and pancreatic ductal fluid. The markers are selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF) double-positive EV extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF double-positive EV extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles and IF double-positive EV extracellular vesicles.

[0050] For certain methods of treating pancreatic cancer (such as pancreatic ductal adenocarcinoma, PDAC) in a patient, the method includes obtaining a first sample from the patient before neoadjuvant therapy; quantitatively determining the concentration of one or more selected markers in the first sample; administering neoadjuvant therapy to the patient; and then obtaining a second sample from the patient after the administration of neoadjuvant therapy. The method further includes quantitatively determining the concentration of EVs that are positive for one or more selected markers in the second sample; comparing the quantitatively determined concentrations of EVs that are positive for one or more selected markers between the first sample and the second sample; and when the concentration of EVs that are positive for one or more selected markers in the second sample is reduced compared to the first sample, resecting the patient's pancreatic tumor, thereby treating the patient's pancreatic ductal adenocarcinoma. In some embodiments of treating pancreatic cancer, the method further includes obtaining a third sample from the patient after resecting the patient's pancreatic tumor; quantitatively determining the concentration of EVs that are positive for one or more selected markers in the third sample; and administering adjuvant therapy to the subject when the concentration of EVs that are positive for one or more selected markers in the third sample is increased compared to the second sample. An increased concentration of EVs that are positive for one or more selected markers indicates an increased disease burden. The sample obtained is selected from: plasma, serum and pancreatic ductal fluid. The marker is selected from: (i) one or both of thrombospondin-2 (THBS2) positive extracellular vesicles and macrophage migration inhibitory factor (MIF) double positive EV extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2) positive extracellular vesicles and THBS2 positive extracellular vesicles, or (iii) a combination of ALPPL2 positive extracellular vesicles and MIF double positive EV extracellular vesicles, or (iv) a combination of ALPPL2 positive extracellular vesicles, THBS2 positive extracellular vesicles and IF double positive EV extracellular vesicles.

[0051] In yet another method of treating pancreatic cancer in a patient, the method includes obtaining a first sample from the patient; quantitatively determining the concentration of one or more selected markers in the sample, the markers being selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF) extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles, and IF extracellular vesicles; comparing the quantitatively determined concentration of the one or more selected markers with a healthy control value or the patient's previous concentration of the selected marker; assessing the extent of pancreatic cancer in the patient after neoadjuvant therapy using a chest, abdominal, and pelvic computed tomography (CT) or ultrasound of the patient to determine a surgical plan for the patient; and resecting the pancreatic tumor in the patient. The samples obtained were selected from: plasma, serum and pancreatic ductal fluid.

[0052] For the above method, extracellular vesicles that are positive for THBS2, ALPPL2 and / or MIF can be separated from serum, plasma or pancreatic ductal fluid using methods known in the art. In some embodiments, the separation / purification of EVs is not required for the ExoView assay. In certain embodiments, the sample is centrifuged at 10,000g for 15min. In certain embodiments, extracellular vesicles are separated from liquid samples by differential centrifugation according to standard protocols. For example, dead cells are removed by centrifugation at 2,000xg for 10min, and large vesicles and apoptotic bodies are removed by centrifugation at 10,000xg for 30min. Extracellular vesicles can also be separated from liquid samples using commercially available separation kits. In some embodiments, liquid samples from patients can be diluted (e.g., at a ratio of 1:1 to 1:100).

[0053] In other aspects, the disclosure includes a test kit for monitoring the appearance, elimination, regression or progress of a disease in a patient diagnosed with pancreatic cancer or receiving pancreatic cancer screening. The test kit may include one or more of THBS2, ALLPL2 and MIF for testing. In some embodiments, suitable equipment for performing one or more tests and / or instructions for performing the test may be included.

[0054] The above features and disclosure will be further understood from the following claims.

[0055] Example

[0056] 1. Identification of novel pancreatic tumor markers

[0057] Longitudinal samples were obtained from CA19-9 secretors and non-secreters (305 samples in total). The expression of placental alkaline phosphatase-like protein 2 (ALPPL2), thrombospondin-2 (THBS2), and macrophage migration inhibitory factor (MIF) in extracellular vesicles (EVs) was monitored.

[0058] The concentration of marker-positive EVs was determined by Unchained Labs using the ExoView R100 instrument. In brief, serum samples were diluted 1:20 in PBS buffer and loaded onto the EV-TETRA-P chip of NanoView Biosciences (50 μL diluted samples per chip). The EV-TETRA-P chip contains antibodies against EV-specific proteins CD9, CD63, and CD81, which capture EVs onto the chip. Then, the EVs captured on the chip were detected by fluorescently labeled antibodies against THSB2, ALPPL2, and MIF. Images of fluorescently labeled EVs captured on the chip were collected by ExoView R100. Then, the number of EVs positive for a single protein marker or a combination of markers was quantified using the ExoView Analyzer software. The number of particles (EVs) reported in the figure is the average of three captures (CD9, CD81, and CD63) in 50 nanoliters (nL) of undiluted samples.

[0059] Figure 1A and Figure 1B Results representing biomarker-positive EV concentrations in healthy controls and CA19-9 non-secretor and secretor patients. Figure 1A Biomarker-positive EV concentrations for ALPPL2 and THBS2 in healthy controls and patients (CA19-9 non-secretors or secretors) are shown. Figure 1B The concentration of MIF-positive EVs in healthy controls and patients (CA19-9 non-secretors or secretors) and the concentration of serum CA19-9 in patients (CA19-9 non-secretors or secretors) are shown. Unlike CA19-9 levels, elevated levels of ALPPL2, THBS2, and MIF were found in marker-positive EVs in CA19-9 secretors and non-secretors. Therefore, such elevated concentrations of MIF-positive EVs, ALPPL2-positive EVs, THBS2-positive EVs in serum samples indicate the possibility of all types of pancreatic cancer.

[0060] Figure 2A and Figure 2B Represents the concentration of biomarker-positive EVs before and after treatment in CA19-9 secretors. Figure 2AShown are the concentrations of biomarker-positive EVs for ALPPL2 and THBS2 before and after treatment in CA19-9 secretors. Figure 2B The concentration of MIF-positive EVs and serum CA19-9 levels before and after treatment in CA19-9 secretors are shown. The changes in biomarker-positive EV concentrations before and after treatment are consistent with the changes in serum CA19-9 in these patients. The upper graph shows the EV concentrations on a logarithmic scale (Y axis) and the lower graph shows the EV concentrations on an arithmetic scale.

[0061] Figure 3A and Figure 3B Represents the concentration of biomarker-positive EVs in patients with normalized CA19-9 after treatment. Figure 3A Shown are the concentrations of biomarker-positive EVs for ALPPL2 and THBS2 in patients with normalization of CA19-9 after treatment. Figure 3B The concentration of MIF-positive EVs and serum CA19-9 in patients with normalized CA19-9 after treatment are shown. The changes in biomarker-positive EV concentrations before and after treatment are consistent with the changes in serum CA19-9 in these patients. The upper graph shows the EV concentrations on a logarithmic scale (Y axis) and the lower graph shows the EV concentrations on an arithmetic scale.

[0062] Figure 4A and Figure 4B Represents the concentration of biomarker-positive EVs before and after treatment in CA19-9 non-secretors. Figure 4A Shown are the concentrations of biomarker-positive EVs for ALPPL2 and THBS2 before and after treatment in CA19-9 non-secretors. Figure 4B MIF-positive EV concentrations and serum CA19-9 concentrations before and after treatment in CA19-9 non-secretors are shown.

[0063] FIG. 5A to FIG. 5C Baseline and pre- and post-treatment results for the combination of THSB2 and MIF in healthy controls and CA19-9 secretors and non-secretors are shown. Figure 5A Baseline levels of THSB2 / MIF double-positive EV concentrations in healthy controls and patients (CA19-9 non-secretors or secretors) are shown. Figure 5B The concentration of THSB2 / MIF double-positive EVs before and after treatment in patients (CA19-9 secretors) is shown (secretors). Figure 5C The concentration of THSB2 / MIF double-positive EVs before and after treatment in patients (CA19-9 non-secretors) is shown.

[0064] Fig. 6A , Figure 6B and Figure 6C It was shown that a combination of 2 or 3 markers can effectively monitor disease burden in PDAC patients. Fig. 6A It was shown that the dual combination (double positivity) of MIF / APPL2 or ALLP2 / THBS2 can effectively monitor the disease burden in PDAC patients. Figure 6B It was shown that the triple combination of MIF, ALPPL2, and THBS2 (triple positivity) can effectively monitor the disease burden in PDAC patients. Figure 6C It was shown that the dual combination of AlPPL2 and THBS2 can effectively monitor the disease burden in PDAC patients (CA19-9 secretors).In some embodiments, the measurement results of EVs are double or triple positive for the markers, rather than combining the measurement results of single marker positive EVs of different markers.

[0065] Therefore, tracking the concentrations of MIF-positive EVs, ALPPL2-positive EVs, and THBS2-positive EVs, alone or in combination, in fluid samples collected from patients can monitor disease burden and treatment effectiveness.

[0066] 2. The concentration of certain EVs is associated with pancreatic tumor size

[0067] FIG. 7A to FIG. 7D A graph showing the correlation between various markers and tumor size as determined according to the Response Evaluation Criteria in Solid Tumors (RECIST) in individual patients who are CA19-9 secretors. In these examples, the markers are ALPP2, THBS2, MIF, and CA19-9.

[0068] FIG. 8A to FIG. 8C A correlation graph showing the correlation between markers and tumor size (RECIST) in individual patients who are non-secretors of CA19-9. The markers shown are ALPP2, THBS2 and MIF. The correlation results between EV markers and tumor size are shown in Table 1.

[0069] Table 1: Correlation between tumor size and EV markers used

[0070]

[0071]

[0072] *Data have been log-transformed.

[0073] The protein-based biomarkers disclosed herein (ALLP2, THBS2 and MIF) alone or in any combination of two or three are effective in monitoring the detection, progression or regression of disease burden (and changes in tumor size or relative size) in patients suffering from pancreatic cancer.

Claims

1. A method for diagnosing pancreatic cancer or monitoring disease burden in a patient diagnosed with pancreatic cancer, the method comprising: a) obtaining a sample from the patient, wherein the sample is selected from the group consisting of plasma, serum and pancreatic ductal fluid; b) quantitatively determining the concentration of one or more selected markers in the sample: the markers are selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF) extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF-positive extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles and IF-positive extracellular vesicles; c) comparing the quantitatively determined concentration of the one or more selected markers to a healthy control value of the selected marker or to a previous concentration of the patient; as well as d) diagnosing the presence or absence of pancreatic cancer based on the comparison of step c), or monitoring the disease burden in the patient, wherein a concentration higher than the healthy control value or the patient's previous concentration indicates pancreatic cancer or increased disease burden.

2. A method of treating pancreatic ductal adenocarcinoma in a patient, the method comprising: a) obtaining a first sample from the patient prior to neoadjuvant therapy, wherein the sample is selected from the group consisting of: plasma, serum, and pancreatic ductal fluid; b) quantitatively determining the concentration of one or more selected markers in the first sample, the markers being selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF) double-positive EV extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF double-positive EV extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles and IF-positive extracellular vesicles; c) administering neoadjuvant therapy to said patient; d) obtaining a second sample from the patient after administration of the neoadjuvant therapy; e) quantitatively determining the concentration of EVs positive for the one or more selected markers in the second sample; f) comparing the quantitatively determined concentration of EVs positive for the one or more selected markers between the first sample and the second sample; and g) resecting the pancreatic tumor in the patient when the concentration of the one or more selected markers is reduced in the second sample compared to the first sample, thereby treating pancreatic ductal adenocarcinoma in the patient.

3. The method according to claim 2, further comprising: h) obtaining a third sample from said patient after resection of a pancreatic tumor from said patient; i) quantitatively determining the concentration of the one or more selected markers in the third sample; as well as j) administering adjuvant therapy to the subject when the concentration of the one or more selected markers is elevated in the third sample compared to the second sample, wherein an increased concentration of the one or more selected markers is indicative of an increased disease burden.

4. A method of treating pancreatic cancer in a patient, the method comprising: a) obtaining a sample from the patient, wherein the sample is selected from the group consisting of plasma, serum and pancreatic ductal fluid; b) quantitatively determining the concentration of one or more selected markers in the sample: the markers are selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF)-positive extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF-positive extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles and IF-positive extracellular vesicles; c) comparing the quantitatively determined concentration of the one or more selected markers to a healthy control value of the selected marker or to a previous concentration of the patient; d) administering neoadjuvant therapy to said patient; e) evaluating the extent of pancreatic cancer in the patient using computed tomography (CT) or ultrasound of the patient's chest, abdomen, and pelvis following neoadjuvant therapy to determine a surgical plan for the patient; and e) resecting the pancreatic tumor from said patient.

5. The method according to any one of claims 2-4, wherein the sample is obtained from plasma, serum or pancreatic ductal fluid of the patient.

6. The method according to any one of claims 1 to 5, wherein the markers comprise (a) THBS2 and ALPPL2, (b) MIF and ALPPL2, (c) MIF and THBS2, or (d) THBS2, ALPPL2 and MIF.

7. The method of claim 6, wherein the markers comprise THBS2, ALLPL2 and MIF.

8. The method according to any one of claims 1-5, wherein the marker comprises THBS2.

9. The method of any one of claims 1-5, wherein the marker comprises MIF.

10. The method of any one of claims 1-9, further comprising correlating the amount of the marker with changes in tumor size associated with pancreatic cancer.

11. The method according to any one of claims 1-10, wherein the patient does not secrete CA19-9.

12. The method according to any one of claims 5 to 11, wherein the antibody is a fluorescently labeled antibody against the selected marker.

13. A method of monitoring the appearance, disappearance, regression or progression of pancreatic cancer in a patient diagnosed with or being screened for pancreatic cancer, the method comprising: a) obtaining a sample from the patient, wherein the sample is selected from the group consisting of plasma, serum and pancreatic ductal fluid; b) quantitatively determining the concentration of EVs in the sample that are positive for one or more selected markers, the markers selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF)-positive extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF-positive extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles, and IF-positive extracellular vesicles; c) comparing the quantitatively determined concentration of the one or more selected markers to a healthy control value of the selected marker or to a previous concentration of the patient; as well as d) monitoring the appearance, disappearance, regression or progression of the disease in a patient diagnosed with or undergoing screening for pancreatic cancer based on the comparison in step c), wherein a concentration higher than the healthy control value or the patient's previous concentration is indicative of the appearance or progression of pancreatic cancer.

14. The method of claim 13, wherein the concentration of EVs positive for one or more selected markers is determined by detecting EVs positive for the one or more selected markers using antibodies against the selected markers, and determining the amount of EVs positive for the one or more selected markers.

15. The method of claim 13 or claim 14, wherein the quantitative determination uses one, two or three markers.

16. The method of any one of claims 13-15, wherein the markers comprise (a) a combination of THBS2 and ALPPL2, (b) MIF and ALPPL2, or (c) MIF and THBS2.

17. The method according to any one of claims 13-16, wherein the markers comprise a combination of THBS2, ALLPL2 and MIF.

18. The method of claim 13 or claim 14, wherein the marker comprises THBS2.

19. The method of claim 13 or claim 14, wherein the marker comprises ALPPL2.

20. The method of claim 13 or claim 14, wherein the marker comprises MIF.

21. The method of any one of claims 13-20, further comprising correlating the amount of the marker with changes in tumor size associated with pancreatic cancer.

22. The method of any one of claims 13-21, wherein the patient does not secrete CA19-9.

23. The method of any one of claims 13-22, wherein the antibody is a fluorescently labeled antibody against the selected marker.

24. A method of determining the duration of pancreatic cancer treatment or the dosage of a pancreatic cancer therapeutic agent, the method comprising: (a) quantitatively determining the amount of one or more markers selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF)-positive extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF-positive extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles and IF-positive extracellular vesicles; as well as (b) correlating the amount of the one or more selected markers with the effectiveness of the treatment by comparing the amount of the one or more selected markers to a healthy control value or a previous concentration of the selected marker in the patient.

25. The method of claim 24, wherein the concentration of EVs positive for one or more selected markers is determined by detecting EVs positive for the one or more selected markers using antibodies against the selected markers, and determining the amount of EVs positive for the one or more selected markers.

26. The method of claim 24 or claim 25, wherein the one or more markers comprise (a) THBS2 and ALPPL2, (b) MIF and ALPPL2, (c) MIF and THBS2, (d) a combination of THBS2, ALLPL2 and MIF, or (e) THBS2, ALPPL2 or MIF.

27. The method of any one of claims 24-26, wherein the patient does not secrete CA19-9.

28. The method of any one of claims 25-27, wherein the antibody is a fluorescently labeled antibody against the selected marker.

29. Use of one or more markers in diagnosing pancreatic cancer, wherein the markers are selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF)-positive extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF-positive extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles and MIF-positive extracellular vesicles.

30. Use of one or more markers for surgical treatment of pancreatic cancer, wherein the markers are selected from: (i) one or both of thrombospondin-2 (THBS2)-positive extracellular vesicles and macrophage migration inhibitory factor (MIF)-positive extracellular vesicles, or (ii) a combination of placental alkaline phosphatase-like protein 2 (ALPPL2)-positive extracellular vesicles and THBS2-positive extracellular vesicles, or (iii) a combination of ALPPL2-positive extracellular vesicles and MIF-positive extracellular vesicles, or (iv) a combination of ALPPL2-positive extracellular vesicles, THBS2-positive extracellular vesicles and MIF-positive extracellular vesicles.

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